Ascorbic acid is a very good antioxidant found at high levels in many tissues, including ocular tissues, in comparison to plasma levels. It is involved in many physiological chemical reactions in which it is subsequently oxidized to a compound known as dehydroascorbic acid (DHAA) which, if allowed to accumulate, may be toxic. In normal, healthy tissues, DHAA generally appears to be recycled back to ascorbic acid. The suggestion has been made that the maintenance of a high ratio of ascorbic acid to DHAA in a particular tissue may be an indication of the health of that tissue. A recycling between the oxidized and reduced forms of the vitamin is absolutely necessary for maintaining a normally high ratio. The physiological mechanisms for recycling and the consequences of impaired recycling are not clearly understood and may be much more important than has been generally believed. The long term goal of this research project is to explain the overall role of vitamin C in maintaining normal physiological function at the cellular and subcellular level. The investigation will include the development of an understanding of physiological, biochemical, and molecular factors pertaining to specific mechanism(s) of normal vitamin C metabolism, causes of abnormal metabolism observed in stressed conditions, and the consequences of an impaired handling of the vitamin for oxidative and other physiological processes within the cell. Specific aims are to 1) identify and evaluate the enzymatic mechanism(s) by which ascorbic acid is maintained in its useful; nontoxic reduced form in mammalian tissues, including ocular tissues; 2) investigate the significance of processes that influence the steady state ratio of AA/DHAA; and 3) determine the role and regulation of protein-related recycling mechanisms under normal and diabetic (hyperglycemic) conditions.